Ring-Constrained (N)-methanocarba nucleosides as adenosine receptor agonists: independent 5'-uronamide and 2'-deoxy modifications

Novel methanocarba adenosine analogues, having the pseudo-ribose northern (N) conformation preferred at adenosine receptors (ARs), were synthesized and tested in binding assays. The 5'-uronamide modification preserved [N6-(3-iodobenzyl)] or enhanced (N6-methyl) affinity at A3ARs, while the 2'-deoxy modification reduced affinity and efficacy in a functional assay.     

Interaction of nucleotides with Asp(351) and the conserved phosphorylation loop of sarcoplasmic reticulum Ca(2+)-ATPase.

The nucleotide binding properties of mutants with alterations to Asp(351) and four of the other residues in the conserved phosphorylation loop, (351)DKTGTLT(357), of sarcoplasmic reticulum Ca(2+)-ATPase were investigated using an assay based on the 2', 3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP) photolabeling of Lys(492) and competition with ATP. In selected cases where the competition assay showed extremely high affinity, ATP binding was also measured by a direct filtration assay. At pH 8.5 in the absence of Ca(2+), mutations removing the negative charge of Asp(351) (D351N, D351A, and D351T) produced pumps that bound MgTNP-8N(3)-ATP and MgATP with affinities 20-156-fold higher than wild type (K(D) as low as 0.006 microM), whereas the affinity of mutant D351E was comparable with wild type. Mutations K352R, K352Q, T355A, and T357A lowered the affinity for MgATP and MgTNP-8N(3)-ATP 2-1000- and 1-6-fold, respectively, and mutation L356T completely prevented photolabeling of Lys(492). In the absence of Ca(2+), mutants D351N and D351A exhibited the highest nucleotide affinities in the presence of Mg(2+) and at alkaline pH (E1 state). The affinity of mutant D351A for MgATP was extraordinarily high in the presence of Ca(2+) (K(D) = 0.001 microM), suggesting a transition state like configuration at the active site under these conditions. The mutants with reduced ATP affinity, as well as mutants D351N and D351A, exhibited reduced or zero CrATP-induced Ca(2+) occlusion due to defective CrATP binding.     

Dissimilatory Reduction of NO(2) to NH(4) and N(2)O by a Soil Citrobacter sp

Dissimilatory reduction of NO(2) to N(2)O and NH(4) by a soil Citrobacter sp. was studied in an attempt to elucidate the physiological and ecological significance of N(2)O production by this mechanism. In batch cultures with defined media, NO(2) reduction to NH(4) was favored by high glucose and low NO(3) concentrations. Nitrous oxide production was greatest at high glucose and intermediate NO(3) concentrations. With succinate as the energy source, little or no NO(2) was reduced to NH(4) but N(2)O was produced. Resting cell suspensions reduced NO(2) simultaneously to N(2)O and free extracellular NH(4). Chloramphenicol prevented the induction of N(2)O-producing activity. The K(m) for NO(2) reduction to N(2)O was estimated to be 0.9 mM NO(2), yet the apparent K(m) for overall NO(2) reduction was considerably lower, no greater than 0.04 mM NO(2). Activities for N(2)O and NH(4) production increased markedly after depletion of NO(3) from the media. Amendment with NO(3) inhibited N(2)O and NH(4) production by molybdate-grown cells but not by tungstate-grown cells. Sulfite inhibited production of NH(4) but not of N(2)O. In a related experiment, three Escherichia coli mutants lacking NADH-dependent nitrite reductase produced N(2)O at rates equal to the wild type. These observations suggest that N(2)O is produced enzymatically but not by the same enzyme system responsible for dissimilatory reduction of NO(2) to NH(4).     

Ichthyobodo necator (Kinetoplastida)--a complex of sibling species

Ichthyobodo necator is a parasitic flagellate that attacks fishes, causing disease problems in freshwater worldwide. Findings of similar flagellates in strictly marine fishes have indicated that ichthyobodiosis may be caused by more than 1 flagellate species. We obtained partial small subunit rDNA (ssu rDNA) sequences of 14 Ichthyobodo isolates originating from fishes in Norway, Japan, Singapore, South Africa and Brazil, and identified 8 strains or species, including 2 species infecting cultured salmon in Norway. An Ichthyobodo species isolated from the skin of Atlantic salmon parr in freshwater is suggested to represent L. necator sensu stricto, while another species, showing particular affinity for the gills, infects salmon in both fresh- and seawater. Atlantic cod is infected with a marine Ichthyobodo species unrelated to those infecting salmonids; 2 cyprinids originating from different parts of the world had related Ichthyobodo strains/species, and 2 isolates from unrelated North and South American fishes were also closely related. The phylogenetic relationships of the Ichthyobodo isolates is described, and the implications of the molecular findings on past and future morphological studies of Ichthyobodo spp. are discussed.     

Synthesis and biological evaluation of new 4-arylpiperidines and 4-aryl-4-piperidinols: dual Na(+) and Ca(2+) channel blockers with reduced affinity for dopamine D(2) receptors.

A series of novel 4-arylpiperidines and 4-aryl-4-piperidinols (2a-f, 3a-f and 4a-f) was synthesized and evaluated for blocking effects on both neuronal Na(+) and T-type Ca(2+) channels and binding affinity for dopamine D(2) receptors. Most of the compounds blockaded both ion channels with potency greater than or equal to flunarizine 1a which was adopted as a reference standard. In addition, these compounds had significantly reduced affinity for dopamine D(2) receptors which is common in this class of structure. Compounds 2a-f, 3a-f and 4a-f exhibited potent anticonvulsant effects following systemic (ip) administration on audiogenic seizures in DBA/2 mice, indicating their excellent brain permeability. The neuroprotective activity of 2a, 3a and 4a was also assessed in a transient middle cerebral artery occlusion (MCAO) model. These compounds significantly reduced neuronal damage without affecting ischemic hyperthemia, while flunarizine 1a produced only minor reductions. In particular, 4a had 1.7-fold the potency in this MCAO model but only 1/20 the affinity for dopamine D(2) receptors of 1a. The superposition of 2a, 3a and 4a on the basis of analyses of systematic conformation and similar structure has revealed that the cinnamyl, phenacyl and phenoxypropanol groups are likely to be structurally and biologically equivalent. Moreover, the superposition of 2a and 2f shows that diphenyl ether and biphenyl groups occupy a similar space, suggesting that both groups act as a bioisostere for the blockade of ion channels; however, this is not the case for dopamine D(2) receptors since only biphenyl compounds such as 2f had high affinity similar to flunarizine 1a. Compound 4a (SUN N5030) has a good pharmacological profile and may be useful in the alleviation and treatment of ischemic diseases.     

Heterogeneity of the Type 3 copper in Japanese-lacquer-tree (Rhus vernicifera) laccase.

The two steps of the titration of the Japanese-lacquer-tree (Rhus vernicifera) laccase with N3- [Morpurgo, Rotilio, Finazzi-Agrò & Mondovi (1974) Biochim. Biophys. Acta 336, 324-328; LuBien, Winkler, Thamann, Scott, Co, Hodgson & Solomon (1981) J. Am. Chem. Soc. 103, 7014-7016] were shown to be two distinct reactions, each involving one different portion of the native enzyme molecules. The difference consists in the oxidation state of the Type 3 Cu, which is reduced in the portion with higher affinity for N3- and oxidized in the portion with lower affinity for N3-. The difference is eliminated by treatment with oxidizing (H2O2) or reducing agents, and a single N3- adduct is then formed. The e.p.r. spectra of the H2O2-treated enzyme and of its F- derivatives support this interpretation of the results. The similarity of the spectroscopic properties of the high-affinity N3- adduct to those of the N3- adducts of half-met-haemocyanins and half-met-tyrosinase is discussed.     

2-(N-acyl) and 2-N-acyl-N(6)-substituted analogues of adenosine and their affinity at the human adenosine receptors

A series of 2-(N-acyl) and 2-(N-acyl)-N(6)-alkyladenosine analogues have been synthesized from the intermediate 2-amino-6-chloroadenosine derivatives (2b and 7) and evaluated for their affinity at the human A(1), A(2A), and A(3) receptors. We found that 2-(N-acyl) derivatives of adenosine showed relatively low affinity at A(2A) and A(3) receptors, while the N(6)-cyclopentyl substituent in 4h and 4i induced high potency [A(1) (K(i))=20.7 and 31.8 nM respectively] at the A(1) receptor and resulted therefore in increased selectivity for this subtype. The general synthetic methods and their binding studies are presented herein.     

Reciprocal mutations of highly conserved residues in transmembrane helices 2 and 7 of the alpha(2A)-adrenoceptor restore agonist activation of G(i1)alpha.

Fusion proteins were constructed between the alpha(2A)-adrenoceptor and the alpha-subunit of the G-protein G(i1). Mutation of the highly conserved Asp(79) in transmembrane (TM) helix 2 of the receptor to Asn reduced the capacity of agonists to activate G(i1)alpha by 95% without altering [3H]antagonist or agonist ligand-binding affinity. A reciprocal mutation in TM helix 7 (Asn(422)Asp) was without effect on signalling effectiveness. Combination of these two mutations overcame the effect of the Asp(79)Asp mutation. By examining alterations in this helix 2-helix 7 microdomain, we further demonstrate the utility of receptor-G-protein fusion proteins to quantitate mutational effects on receptor-G-protein interactions and information transfer.     

Integrin alpha(2)I domain recognizes type I and type IV collagens by different mechanisms

The collagens are recognized by the alphaI domains of the collagen receptor integrins. A common structural feature in the collagen-binding alphaI domains is the presence of an extra helix, named helix alphaC. However, its participation in collagen binding has not been shown. Here, we have deleted the helix alphaC in the alpha(2)I domain and tested the function of the resultant recombinant protein (DeltaalphaCalpha(2)I) by using a real-time biosensor. The DeltaalphaCalpha(2)I domain had reduced affinity for type I collagen (430 +/- 90 nM) when compared with wild-type alpha(2)I domain (90 +/- 30 nM), indicating both the importance of helix alphaC in type I collagen binding and that the collagen binding surface in alpha(2)I domain is located near the metal ion-dependent adhesion site. Previous studies have suggested that the charged amino acid residues, surrounding the metal ion-dependent adhesion site but not interacting with Mg(2+), may play an important role in the recognition of type I collagen. Direct evidence indicating the participation of these residues in collagen recognition has been missing. To test this idea, we produced a set of recombinant alpha(2)I domains with mutations, namely D219A, D219N, D219R, E256Q, D259N, D292N, and E299Q. Mutations in amino acids Asp(219), Asp(259), Asp(292), and Glu(299) resulted in weakened affinity for type I collagen. When alpha(2) D219N and D292N mutations were introduced separately into alpha(2)beta(1) integrin expressed on Chinese hamster ovary cells, no alterations in the cell spreading on type I collagen were detected. However, Chinese hamster ovary cells expressing double mutated alpha(2) D219N/D292N integrin showed remarkably slower spreading on type I collagen, while spreading on type IV collagen was not affected. The data indicate that alpha(2)I domain binds to type I collagen with a different mechanism than to type IV collagen.     

Anion permeation in Ca(2+)-activated Cl(-) channels

Ca(2+)-activated Cl channels (Cl(Ca)Cs) are an important class of anion channels that are opened by increases in cytosolic [Ca(2+)]. Here, we examine the mechanisms of anion permeation through Cl(Ca)Cs from Xenopus oocytes in excised inside-out and outside-out patches. Cl(Ca)Cs exhibited moderate selectivity for Cl over Na: P(Na)/P(Cl) = 0.1. The apparent affinity of Cl(Ca)Cs for Cl was low: K(d) = 73 mM. The channel had an estimated pore diameter >0.6 nm. The relative permeabilities measured under bi-ionic conditions by changes in E(rev) were as follows: C(CN)(3) > SCN > N(CN)(2) > ClO(4) > I > N(3) > Br > Cl > formate > HCO(3) > acetate = F > gluconate. The conductance sequence was as follows: N(3) > Br > Cl > N(CN)(2) > I > SCN > COOH > ClO(4) > acetate > HCO(3) = C(CN)(3) > gluconate. Permeant anions block in a voltage-dependent manner with the following affinities: C(CN)(3) > SCN = ClO(4) > N(CN)(2) > I > N(3) > Br > HCO(3) > Cl > gluconate > formate > acetate. Although these data suggest that anionic selectivity is determined by ionic hydration energy, other factors contribute, because the energy barrier for permeation is exponentially related to anion hydration energy. Cl(Ca)Cs exhibit weak anomalous mole fraction behavior, implying that the channel may be a multi-ion pore, but that ions interact weakly in the pore. The affinity of the channel for Ca(2+) depended on the permeant anion at low [Ca(2+)] (100-500 nM). Apparently, occupancy of the pore by a permeant anion increased the affinity of the channel for Ca(2+). The current was strongly dependent on pH. Increasing pH on the cytoplasmic side decreased the inward current, whereas increasing pH on the external side decreased the outward current. In both cases, the apparent pKa was voltage-dependent with apparent pKa at 0 mV = approximately 9.2. The channel may be blocked by OH(-) ions, or protons may titrate a site in the pore necessary for ion permeation. These data demonstrate that the permeation properties of Cl(Ca)Cs are different from those of CFTR or ClC-1, and provide insights into the nature of the Cl(Ca)C pore.     

Exploring distal regions of the A3 adenosine receptor binding site: sterically constrained N6-(2-phenylethyl)adenosine derivatives as potent ligands

We synthesized phenyl ring-substituted analogues of N(6)-(1S,2R)-(2-phenyl-1-cyclopropyl)adenosine, which is highly potent in binding to the human A(3)AR with a Ki value of 0.63 nM. The effects of these structural changes on affinity at human and rat adenosine receptors and on intrinsic efficacy at the hA(3)AR were measured. A 3-nitrophenyl analogue was resolved chromatographically into pure diastereomers, which displayed 10-fold stereoselectivity in A(3)AR binding in favor of the 1S,2R isomer. A molecular model defined a hydrophobic region (Phe168) in the putative A(3)AR binding site around the phenyl moiety. A heteroaromatic group (3-thienyl) could substitute for the phenyl moiety with retention of high affinity of A(3)AR binding. Other related N(6)-substituted adenosine derivatives were included for comparison. Although the N(6)-(2-phenyl-1-cyclopropyl) derivatives were full A(3)AR agonists, several other derivatives had greatly reduced efficacy. N(6)-Cyclopropyladenosine was an A(3)AR antagonist, and adding either one or two phenyl rings at the 2-position of the cyclopropyl moiety restored efficacy. N(6)-(2,2-Diphenylethyl)adenosine was an A(3)AR antagonist, and either adding a bond between the two phenyl rings (N(6)-9-fluorenylmethyl) or shortening the ethyl moiety (N(6)-diphenylmethyl) restored efficacy. A QSAR study of the N(6) region provided a model that was complementary to the putative A(3)AR binding site in a rhodopsin-based homology model. Thus, a new series of high-affinity A(3)AR agonists and related nucleoside antagonists was explored through both empirical and theoretical approaches.     

Identification of alpha 2-adrenergic receptor sites in human retinoblastoma (Y-79) and neuroblastoma (SH-SY5Y) cells

The existence of specific alpha 2-adrenergic receptor sites has been shown in human retinoblastoma (Y-79) and neuroblastoma (SH-SH5Y) cells using direct radioligand binding. [3H]Rauwolscine, a selective alpha 2-adrenergic receptor antagonist, exhibited high affinity, saturable binding to both Y-79 and SH-SY5Y cell membranes. The binding of alpha 1 specific antagonist, [3H]Prazocine, was not detectable in either cell type. Competition studies with antagonists yielded pharmacological characteristics typical of alpha 2-adrenergic receptors: rauwolscine greater than yohimbine greater than phentolamine greater than prazocine. Based on the affinity constants of prazocine and oxymetazoline, it appears that Y-79 cells contain alpha 2A receptor, whereas SH-SY5Y cells probably represent a mixture of alpha 2A and alpha 2B receptors. alpha 2-agonists clonidine and (-)epinephrine inhibition curves yielded high and low affinity states of the receptor in SH-SY5Y cells. Gpp(NH)p and sodium ions reduced the proportion of high affinity sites of alpha 2 receptors. These two neuronal cell lines of human origin would prove useful in elucidating the action and regulation of human alpha 2-adrenergic receptors and their interaction with other receptor systems.     

Sub-Parts-Per-Billion Nitrate Method: Use of an N(2)O-Producing Denitrifier to Convert NO(3) or NO(3) to N(2)O

A more sensitive analytical method for NO(3) was developed based on the conversion of NO(3) to N(2)O by a denitrifier that could not reduce N(2)O further. The improved detectability resulted from the high sensitivity of the Ni electron capture gas chromatographic detector for N(2)O and the purification of the nitrogen afforded by the transformation of the N to a gaseous product with a low atmospheric background. The selected denitrifier quantitatively converted NO(3) to N(2)O within 10 min. The optimum measurement range was from 0.5 to 50 ppb (50 mug/liter) of NO(3) N, and the detection limit was 0.2 ppb of N. The values measured by the denitrifier method compared well with those measured by the high-pressure liquid chromatographic UV method above 2 ppb of N, which is the detection limit of the latter method. It should be possible to analyze all types of samples for nitrate, except those with inhibiting substances, by this method. To illustrate the use of the denitrifier method, NO(3) concentrations of <2 ppb of NO(3) N were measured in distilled and deionized purified water samples and in anaerobic lake water samples, but were not detected at the surface of the sediment. The denitrifier method was also used to measure the atom% of N in NO(3). This method avoids the incomplete reduction and contamination of the NO(3) -N by the NH(4) and N(2) pools which can occur by the conventional method of NO(3) analysis. N(2)O-producing denitrifier strains were also used to measure the apparent K(m) values for NO(3) use by these organisms. Analysis of N(2)O production by use of a progress curve yielded K(m) values of 1.7 and 1.8 muM NO(3) for the two denitrifier strains studied.     

The Functional Differences between Pro-survival and Pro-apoptotic B Cell Lymphoma 2 (Bcl-2) Proteins Depend on Structural Differences in Their Bcl-2 Homology 3 (BH3) Domains

Bcl-2 homology 3 (BH3) domains are short sequence motifs that mediate nearly all protein-protein interactions between B cell lymphoma 2 (Bcl-2) family proteins in the intrinsic apoptotic cell death pathway. These sequences are found on both pro-survival and pro-apoptotic members, although their primary function is believed to be associated with induction of cell death. Here, we identify critical features of the BH3 domains of pro-survival proteins that distinguish them functionally from their pro-apoptotic counterparts. Biochemical and x-ray crystallographic studies demonstrate that these differences reduce the capacity of most pro-survival proteins to form high affinity “BH3-in-groove” complexes that are critical for cell death induction. Switching these residues for the corresponding residues in Bcl-2 homologous antagonist/killer (Bak) increases the binding affinity of isolated BH3 domains for pro-survival proteins; however, their exchange in the context of the parental protein causes rapid proteasomal degradation due to protein destabilization. This is supported by further x-ray crystallographic studies that capture elements of this destabilization in one pro-survival protein, Bcl-w. In pro-apoptotic Bak, we demonstrate that the corresponding distinguishing residues are important for its cell-killing capacity and antagonism by pro-survival proteins.     

Vaccinia virus N1L protein resembles a B cell lymphoma-2 (Bcl-2) family protein

Poxviruses encode immuno-modulatory proteins capable of subverting host defenses. The poxvirus vaccinia expresses a small 14-kDa protein, N1L, that is critical for virulence. We report the crystal structure of N1L, which reveals an unexpected but striking resemblance to host apoptotic regulators of the B cell lymphoma-2 (Bcl-2) family. Although N1L lacks detectable Bcl-2 homology (BH) motifs at the sequence level, we show that N1L binds with high affinity to the BH3 peptides of pro-apoptotic Bcl-2 family proteins in vitro, consistent with a role for N1L in modulating host antiviral defenses.     

Synthesis of 2-(2,3-dimethoxyphenyl)-4-(aminomethyl)imidazole analogues and their binding affinities for dopamine D(2) and D(3) receptors.

A series of 2-(2,3-dimethoxyphenyl)-4-(aminomethyl)imidazole derivatives was prepared and their affinity for dopamine D₂ and D₃ receptors was measured using in vitro binding assays. Several oxadiazole analogues were also prepared and tested for their affinity for dopamine D₂ and D₃ receptors. The results of receptor binding studies indicated that the incorporation of an imidazole moiety between the phenyl ring and the basic nitrogen did not significantly increase the selectivity for dopamine D₃ receptors, whereas the incorporation of an oxadiazole at the same region resulted in a total loss of affinity for both dopamine receptor subtype binding sites. The most selective compound in this series is 2-(5-bromo-2,3-dimethoxyphenyl)-4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolinomethyl)imidazole (5i), which has a D₃ receptor affinity of 21 nM and a 7-fold selectivity for D₃ versus D₂ receptors. The binding affinity for σ₁ and σ₂ receptors was also measured, and the results showed that several analogues were selective σ₁ receptor ligands.     

Functional role of the spatial proximity of Asp114(2.50) in TMH 2 and Asn332(7.49) in TMH 7 of the mu opioid receptor

We examined whether a proposed spatial proximity between Asp114(2.50) and Asn332(7.49) affected the functional properties of the mu opioid receptor. The D114(2.50)N mutant had reduced binding affinities for morphine, DAMGO and CTAP, but not for naloxone and [3H]diprenorphine; this mutation also abolished agonist-induced increase in [35S]GTPgammaS binding. The N332(7.49)D mutation eliminated detectable binding of either [3H]diprenorphine or [3H]DAMGO. The combined D114(2.50)N-N332(7.49)D mutation restored high affinity binding for [3H]diprenorphine, CTAP and naloxone, and restored partially the binding affinities, potencies and efficacies of morphine and DAMGO. Thus, reciprocal mutations of Asp114(2.50) and Asn332(7.49) compensate for the detrimental effects of the single mutations, indicating that the residues are adjacent in space and that their chemical functionalities are important for ligand binding and receptor activation.     

Incorporation of (2S,3S) and (2S,3R) beta-methyl aspartic acid into RGD-containing peptides

We report the synthesis and biological activity of a series of side-chain-constrained RGD peptides containing the (2S,3R) or (2S,3S) beta-methyl aspartic acid within the RGD sequence. These compounds have been assayed for binding to the integrin receptors alpha(IIb)beta3 and alpha(v)beta3 and the results demonstrate the importance of the side-chain orientation of this particular residue within the RGD sequence. Based on our findings, the (2S,3S) beta-methylated analogues of our RGD sequences maintain their binding potency to the integrin receptors while the (2S,3R) beta-methylated analogues exhibit a drastically reduced binding affinity. Our studies demonstrate that the three-dimensional orientation of the aspartyl side chain is a very important parameter for integrin binding and that small changes that affect the side-chain orientations give rise to drastic changes in binding affinity. These results provide important information for the design of more potent RGD mimetics.     

Design of (N)-methanocarba adenosine 5'-uronamides as species-independent A3 receptor-selective agonists

2-Chloro-5′-N-methylcarboxamidoadenosine analogues containing the (N)-methanocarba (bicyclo[3.1.0]hexane) ring system as a ribose substitute display increased selectivity as agonists of the human A₃ adenosine receptor (AR). However, the selectivity in mouse was greatly reduced due to an increased tolerance of this ring system at the mouse A₁AR. Therefore, we varied substituents at the N⁶ and C2 positions in search of compounds that have improved A₃AR selectivity and are species independent. An N⁶-methyl analogue was balanced in affinity at mouse A₁/A₃ARs, with high selectivity in comparison to the A_2AAR. Substitution of the 2-chloro atom with larger and more hydrophobic substituents, such as iodo and alkynyl groups, tended to increase the A₃AR selectivity (up to 430-fold) in mouse and preserve it in human. Extended and chemically functionalized alkynyl chains attached at the C2 position of the purine moiety preserved A₃AR selectivity more effectively than similar chains attached at the 3-position of the N⁶-benzyl group.